The long-term goals of this applicant are to determine the cellular & molecular mechanisms mediating placental angiogenesis & ultimately control placental blood flow and function. We will use Ovine Fetal Placental Artery Endothelial (OFPAE) & Human Placental Microvascular (HPME) cell lines for this purpose. These placental endothelial cells were chosen because dramatic placental vascular growth during human & ovine pregnancy is associated with dramatic increases in umbilical blood flow, which is critical for fetal growth & survival as well as neonatal birth weights and survivability. The angiogenic factors, bFGF & VEGF, are key factors regulating angiogenesis & production of endothelial nitric oxide (NO; a potent vasodilator). Actions of bFGF & VEGF are tightly mediated by proteins kinases (i.e. Akt & p38 MAPK) & protein phosphatases (i.e. PP2A & PP2B). The hypothesis is that in the placenta bFGF- & VEGF-induced angiogenesis is modulated in part via activation of the PI3K/Akt and p38 MAPK signal pathways as well as inhibition of PP2A and PP2B, which in turn increase NO production, so promoting fetal placental angiogenesis. 3 Specific Aims will be addressed using the well-characterized OFPAE cell line. We will determine: AIM I. if bFGF- & VEGF-stimulated cell proliferation & migration are mediated in part via activation of the PI3K/Akt & p38 MAPK pathways by treating cells with bFGF or VEGF in the presence of specific PI3K or p38 MAPK inhibitors, using cell proliferation & migration assays, Western analysis, and kinase activity assays; AIM II. if bFGF & VEGF activate Akt & p38 MAPK in part via inhibiting PP2A & PP2B activities, by treating cells with bFGF or VEGF in the presence of the specific protein phosphatase inhibitors, using Western analysis, protein phosphatase & kinase activity assays, and cell proliferation & migration assays; and AIM III. If bFGF- & VEGF elevate NO production via activation of PI3K/Akt & p38 MAPK as well as inhibition of PP2A & PP2B, regulating eNOS phosphorylation. An additional Specific Aim IV will use the HPME cell line to confirm the key observations made from OFPAE cells. These studies will glean important information on the mechanisms regulating placental angiogenesis.